Therapy of estrogen-associated disorders

ABSTRACT

A method for the treatment, prophylaxis, amelioration or prevention of disorders associated with abnormally high activity of steroidal estrogens, which includes administering to a human subject a composition comprising an isoflavone-containing extract of clover or chick peas, said extract comprising primarily biochanin, or a ration of biochanin to one or more of the isoflavones from the group formononetin, daidzein and genistein in the range of from about 2:1 to about 5:1, optionally in association with one or more pharmaceutically acceptable carriers, excipients, auxiliaries and/or diluents is described. Also described are compositions and uses comprising the isoflavone containing extracts.

This invention relates to methods and compositions for the treatment,prophylaxis, amelioration or prevention of disorders associated withabnormally high activity of steroidal estrogens, using a plant extractenriched for estrogenic isoflavones preferably comprising predominantlybiochanin or a high ratio of biochanin to a mixture of other estrogenicisoflavones comprising formononetin, daidzein and genistein. Thetargeted conditions specifically include uterine fibroids, polycysticovarian disease, ovarian cysts, mastalgia, cyclical acne, endometriosisand endometrial hyperplasia.

The aforementioned conditions usually are considered as a group becauseof a number of common characteristics. They occur almost exclusively inpre-menopausal women and therefore are thought to be associated withhigh blood levels of steroidal sex hormones, and they are associatedwith benign, hyperplastic and proliferative changes in the tissues ofthe female reproductive tract (with the exception of cyclical acne).

The prominent role of steroidal estrogen in these conditions issuggested by the observations firstly that these conditions and symptomsoccur almost predominantly in pre-menopausal women and usually disappearwith the onset of menopause, secondly, they often get worse during themid-phase of the menstrual cycle when cyclical estrogen levels arehighest, they usually are exacerbated by the use of steroidal estrogentherapy, and lastly they usually respond to therapies such as GnrHagonists, oral contraception, or oophorectomy, all of which have theeffect of reducing steroidal estrogen production in the body.

Uterine fibroids are a particularly common problem with more than 30% ofwomen developing fibroids by the time they reach menopause. Fibroids arebenign adenomas originating in the uterine wall and composed of fibroustissue. For some women, the fibroids can be small and essentiallyasymptomatic. For other women, the fibroids can be of such a size tocause symptoms such as severe menstrual bleeding and uterine cramping(with fibroids that disrupt the endometrium) or constipation and urinaryfrequency (with large, space-occupying fibroids that grow out into thepelvic cavity). Estrogen therapy is known to aggravate fibroids and thesymptoms associated with fibroids (Grodstein et al. “Postmenopausalhormone use and colecystectomy in a large prospective study.” Obstetricsand Gynaecology: 1994: 83(1), 5-11; Akkad et al. “Abnormal uterinebleeding on HRT: The importance of intrauterine structuralabnormalities.” Obstetrics and Gynaecology, 1995. 86(3): 330-334; Seneret al. “The effects of HRT on uterine fibroids in postmenopausal women.”Fertility and Sterility, 1996. 65(2): 354-357]. The usual treatment forfibroids is surgical removal (hysterectomy or hysterotomy).

Endometrial hyperplasia is thought to affect between 5-15% ofpre-menopausal women. It involves an abnormal thickening of theendometrium that is not completely shed at the time of menstruation. Thesymptoms can include painful and heavy menstruation, and painful sexualintercourse. Treatment usually consists of surgery (curettage orhysterectomy).

Endometriosis affects about 5% of pre-menopausal women. This conditionis due to the appearance of endometrium in the peritoneal cavity.Patches of endometrium can grow on the serosal surface of the ovaries,uterus, bladder, large intestine or the peritoneum. These patchesrespond to normal hormonal changes over the menstrual cycle in parallelto that of the endometrium in the uterus and can bleed, swell and causesevere pelvic discomfort and pain. Standard therapy of endometriosis issurgical ablation of the abnormal tissue, although the recurrence rateis high, requiring ongoing surgical treatment [Namnoum et al. “Incidenceof symptom recurrence after hysterectomy for endometriosis.” Fertilityand Sterility, 1995. 64(5): 898-902]. Endometriosis also is aggravatedby estrogen therapy [Goh et al. “Postmenopausal endometrioma and HRT”Australia New Zealand of Obstetrics and Gynaecology, 1992. 32(4):384-385].

Ovarian cysts are thought to affect up to about 20% of pre-menopausalwomen. The pathology is that of multiple, incomplete follicles withinthe body of the ovary. A variant of this condition is known aspolycystic ovarian disease which is characterised by excessive androgenproduction from the follicles stimulated by abnormally high insulinlevels. Excess androgens are often converted to steroidal estrogen. Theusual symptoms of polycystic ovarian disease are hirsutism and acne. Thenormal treatment for these conditions are GnrH agonists that function bydown-regulating the release of gonadotrophins (FSH and Li) from thehypothalamus, thereby inhibiting and further ovulation.

Mastalgia is also known as cyclical mastalgia or fibrocystic breastdisease. It is characeterised by the retention of fluid in cysts withinthe fibrous tissue of the breast. It normally is associated withswelling, pain and tenderness, with symptoms usually worsening about themiddle of the menstrual cycle when estradiol levels peak in the blood.No effective therapy for this condition is known.

Cyclical acne normally is restricted to post-adolescent women. It isassociated with severe acne over the face and upper torso and normallythe acne worsens on a cyclical basis in parallel with the menstrualcycle. The normal therapy for this condition is oral contraception inorder to regulate ovulation and estrogen production.

In general, the management of these aforementioned conditions isunsatisfactory. Surgery is the most common method of treatment, andapart from the dramatic and intrusive nature of this approach, aninevitable outcome often is sterility as a result of removal of ovariesand/or uterus. The use of GnrH agonists also is not without adverseconsequences as it invariably leads to premature menopause, with itsattendant increased risks of osteoporosis and heart disease.

The underlying causes of or risk factors for the aforementionedconditions are unknown. Genetic risk factors are not reported, neitherare lifestyle risk factors. There is no reliable epidemiological datareported that links the incidence of any one of these conditions or thegroup of conditions as a whole to specific communities or racial groups,or to lifestyle factors such as diet. However, it generally isrecognised that the conditions or symptoms are associated with excessestrogen stimulation of tissues of the female reproductive tract andthat it would be prudent to avoid any situation likely to aggravateestrogenic activity.

Plant estrogens including estrogenic isoflavones recently have come tomedical attention because they mimic the effect/activity of steroidalestrogens and their biologically active analogues by binding to andactivating estrogen receptors on animals (including human) cells. Plantisoflavones such as formononetin, biochanin; daidzein and genistein areknown to be estrogenic in vitro, acting as agonists for the humanestrogen receptor. Their ability to function as estrogens in the body iswell understood and the epidemiological link between diets high in theseestrogenic isoflavones and low incidences of certain estrogen-deficiencystates also is well documented. This has led to considerable interest inthe use of dietary estrogens such as isoflavones to provide asupplementary estrogenic activity in menopausal women, providing relieffrom estrogen-deficiency symptoms including hot flushes, mood swings,osteoporosis, hypertension, and hypercholesterolaemia.

Given the current understanding about the estrogenic action ofisoflavones, and the adverse consequences of estrogen therapy on theaforementioned pre-menopausal conditions and symptoms, estrogenicisoflavones would appear to be contra-indicated for subjects sufferingfrom those conditions or symptoms. While the incidences of theseconditions are adequately reported in women in Western countries wherethe diet is low in dietary isoflavones, there is no known data on theincidences in communities that typically ingest high levels ofestrogenic isoflavones. It would be reasonable, however, to assume thatit would particularly contra-indicated for pre-menopausal women at riskof the aforementioned conditions and symptoms to extensively consumefoodstuffs such as clover that is the richest source of estrogenicisoflavones in nature. Therefore, the applicants were surprised to findthat isoflavone-containing extracts of clover or chickpeas wereparticularly beneficial in the treatment of estrogen-sensitiveconditions.

Clovers (Trifolium spp.) are one of the richest sources of estrogenicisoflavones in the plant kingdom, with some cultivars containing up to5% of their dry weight as estrogenic isoflavones. The use ofsub-terranean clovers in agriculture is associated with a condition insheep known as ‘clover disease’. This disease is due to excessiveingestion of estrogenic isoflavones resulting in hyperplastic changes inthe lower reproductive tract. The linings of the uterus, cervix andvagina of sheep undergo dramatic proliferative changes and the ovariesshow multiple cyst formation, all of which produces temporary and thenpermanent infertility. The pathology of these changes in sheep closelyparallels that in humans with conditions such as endometrialhyperplasia. The likelihood that estrogenic isoflavones might exacerbatesuch conditions in women, particularly in those women with little priorexposure to isoflavones, is therefore is a real concern.

Clovers such as red clover have enjoyed some therapeutic use by herbalpractitioners over the centuries for various human ailments such asasthma and eczema and have not been noted as having any association withincreased incidence of reproductive disorders. It should be noted thatthe traditional herbal use of red clover employs the flower and thispart of the plant contains very little isoflavone. Also, red cloveroccurs in multiple cultivars and many cultivars have very low isoflavonelevels. Thus it is not possible to ascribe any putative therapeuticbenefit from herbal medicinal use to their isoflavone content, and noris it possible to ascribe any apparent lack of adverse side effects tothe safety of isoflavones.

In spite of this background, the applicant surprisingly has found thatclover extracts preferably those enriched for certain estrogenicisoflavones may be used in the prevention and treatment of conditions ofthe female reproductive tract associated with excessive estrogenactivity. Even more surprising is the finding that a particular ratio ofdifferent types of estrogenic isoflavones in the extract is primarilyresponsible for the demonstrated therapeutic effects. Preferably theextract comprises biochanin as the principal isoflavone. In the contextof this invention, it has been found that the preferred ratio ofbiochanin to the other three main estrogenic isoflavones embraces thevarious naturally-occurring forms of isoflavones including theiraglycone, glycoside, acetyl or malonyl forms.

In accordance with a first aspect of this invention there is provided amethod for the treatment, prophylaxis, amelioration or prevention ofdisorders associated with an abnormally high activity of steroidalestrogen, which includes administering to a human subject a compositioncomprising an isoflavone containing extract of clover or chick pea, oran isoflavone extract of clover or chick pea comprising primarilybiochanin or biochanin and any one or more of the isoflavones from thegroup formononetin, daidzein and genistein at a ratio from about 2:1 toabout 5:1, optionally in association with one or more pharmaceuticallyacceptable carriers, excipients, auxiliaries and/or diluents.

Preferably the disorder is selected from uterine fibroids, polycysticovarian disease, ovarian cysts, cyclical acne, mastalgia, endometriosisand endometrial hyperplasia.

By reference to an extract comprising “primarily biochanin” is meant abiochanin content of at least 65% by weight isoflavone content, such as70 to 100%, more preferably 75 to 90% biochanin, the remainder ofisoflavone content comprising essentially formononetin, daidzein andgenistein.

The high ratio of biochanin to the other three estrogenic isoflavones inthe range from about 2:1 to about 5:1 is responsible for the optimalunexpected therapeutic effects according to this invention. Outside ofthis range of ratios, the therapeutic benefits effectively are notobserved, or if observed are considerably less effective. The ratio ofbiochanin to the other three isoflavones may, for example, be from about7:3 to about 5:1.

In a further aspect of this invention there is provided a compositioncomprising an isoflavone-rich extract from clover or chick pea, saidextract comprising a ratio of biochanin to formononetin, daidzein andgenistein at a ratio from about 2:1 to about 5:1, optionally inassociation with one or more pharmaceutically acceptable carriers,excipients, auxiliaries, and/or diluents.

In a further aspect of the invention there is provided use of acomposition comprising an isoflavone extract of clover or chick pea,said extract comprising primarily biochanin, or a ratio of biochanin toformonentin, daidzein and genistein at a ratio of from about 2:1 toabout 5:1, optionally in association with one or more pharmaceuticallyacceptable carriers, excipients, auxiliaries, and/or diluents for themanufacture of a medicament for the treatment, prophylaxis, ameliorationor prevention of disorders associated with an abnormally high activityof steroidal estrogen.

Scientific interest in isoflavones up until now has focused on daidzeinand genistein. This is in part because of the belief that these twoisoflavones are the most prominent in the human diet. But mainly it isbecause of the general belief that formononetin and biochanin-areunlikely to have any biological significance in their own right.Formononetin and biochanin are methylated versions of daidzein andgenistein respectively and it is thought that humans have the capacityto convert formononetin to daidzein and biochanin to genistein. Insheep, the only animal species in which isoflavone metabolism has beenextensively studied, this conversion is virtually 100% effective. Sheepconsume predominantly formononetin and biochanin as a result of eatingisoflavone-rich clovers, with virtually all formononetin beingdemethylated to daidzein and all biochanin being demethylated togenistein. Reports of analyses of blood or urine of humans ingestingdiets rich in isoflavones has failed to demonstrate any formononetin andbiochanin as being present, leading to the general assumption that thesame situation applies in humans. That is, even if formononetin andbiochanin are present in the diet, there is a general assumption thatthey are converted to daidzein and genistein and that it is these twoisoflavones that hold the true source of biological significance tohumans. The dominant use of soya as a source of dietary isoflavones bythe health food and pharmaceutical industries is testament to this, withsoya containing predominantly daidzein and genistein (95-99%) and onlytrace amounts of biochanin and formononetin.

The inventors were therefore very surprised to find that an extract ofred clover or chick peas, preferably enriched for or comprisingbiochanin as the principal isoflavone, provided a therapeutic effectquite distinct from that seen with the same dose of isoflavonescontaining predominantly daidzein and genistein. This suggests thatbiochanin provides a distinctive and individualistic biological effect.

It has been recognized for some time that the four estrogenicisoflavones have an individual biological profile. That is, genistein isapproximately ten fold more estrogenic than daidzein, and genistein haspotent anti cancer actions while daidzein has virtually none. Thefinding that biochanin might therefore also have a distinctivebiological profile is not surprising, but that it should be able tomaintain this distinction when its presence in the human body has notpreviously been demonstrated is surprising.

This finding was even more surprising given what generally is knownabout the action of isoflavones at the cellular level and the manner inwhich they function as estrogens. Compounds can exert an estrogeniceffect in the body in one of two ways, either indirectly, by stimulatingthe brain to produce gonadotropins that in turn cause the body to makemore estradiol, or directly, by attaching to and activating the humanestrogen receptor (hER). The isoflavones are thought to act directly inthe same way as the steroidal estrogen, estradiol, and some evidenceexists to support that belief. Recent studies, however, point toisoflavones having an important difference to that of estradiol in theway they art as estrogens. Two distinct types of hER now have beenidentified, known as alpha-ER and beta-ER. Whereas estradiol binds withsimilar strengths to both ER types and appears to be able to stimulateboth receptor types equally, the isoflavonoids are quite different. Theyshow significantly greater agonism to the beta-ER than to the alpha-ER.The significance of this phenomenon lies in the fact that alpha-ERdominates on tissues of the female reproductive tract (vagina,endometrium, breast, ovary), while beta-ER dominates on most othertissues (brain, bone, endothelium etc.). The fact that isoflavones haveweak affinity for alpha-ER is confirmed by clinical studies andepidemiological observations that diets rich in estrogenic isoflavoneshave little or no stimulatory effect on female reproductive tissue inpost-menopausal women. But the experience with ‘Clover. Disease’ insheep teaches us that this most likely is a dose-related effect—ifsufficient isoflavones are ingested, then it is possible to achievestimulation of the alpha-ER, producing proliferation of the reproductivetissues. Importantly, however, at normal dietary dosages, estrogenicisoflavones appear to have little or no impact on tissues expressingmainly alpha-ER.

Without wishing to be bound by any particular theory of action, thereare multiple possible mechanisms by which biochanin may be providing atherapeutic benefit in reducing the impact of steroidal estrogens on thereproductive tissues. One likely explanation is the way in whichbiochanin interacts with the alpha-ER, but this again is surprisinggiven that there is no known difference in the way in which any of thefour estrogenic isoflavones behave as ligands with this receptor.

It has been theorised that one of the biological effects of isoflavonesis their ability to ‘competitively inhibit’ the action of steroidalestrogens such as estradiol. By this process, the isoflavonoid, being aweaker estrogen, once bound to the hER and occupying the receptor site,prevents the more estrogenic estradiol reaching the receptor.Isoflavones are weak estrogens, having an estrogenic potency betweenabout 1:250 to 1:2,000 that of estradiol, and while they haveconsiderably lower affinity for the hER compared to estradiol, thesubstantially greater numbers of isoflavones in the blood relative toestradiol means that a significant proportion of hER will be occupied byisoflavones. This theory holds that even though the isoflavonoidproduces a weak estrogenic response, the fact that it has prevented amore potent response by estradiol means that the net effect has been oneof a reduced estrogenic effect. This theory could be used to explain theability of isoflavones to reduce the impact of estradiol on the femalereproductive tissues during the pre-menopausal years when estradiollevels are at their highest.

Biochanin has about the same estrogenic potency as daidzein and there issome evidence that it has lower binding affinity for the hER compared todaidzein. Therefore eves if competitive-inhibition is a contributingfactor the clinical observation by the inventors, then it is even moresurprising that an extract principally comprising biochanin should bemore efficacious than an extract principally comprising daidzein andgenistein.

Preferably the extract of clover is prepared by a water/water-miscibleorganic solvent extract of clover. The ratio of water to water-miscibleorganic solvent is generally in the range of 1:10 to 10:1 and may forexample comprise equal proportions of water and solvent or from 1% to30% (v/v) water-miscible organic solvent. Any water-miscible organicsolvent or a mixture of such solvents may be used. The water-miscibleorganic solvent is preferably a C2-10, more preferably C14water-miscible organic solvent (such as methanol, ethanol, propanol,propylene glycol, erythrite, butanol, butanediol, acetonitrile,ethyleneglycol, ethylacetate, glycidol, glycerol dihydroxyacetone oracetone). The extract in this regard is prepared by exposing the plantmaterial to the water/water-miscible solvent mix. Optionally the mixturemay include an enzyme, which cleaves isoflavone glycosides to theaglucone form. The mixture may be vigorously agitated so as to form anemulsion. The temperature of the mix may range, for example, from anambient temperature to boiling temperature. Exposure time may be betweenone hour to several weeks. One convenient extraction period istwenty-four hours at 90 C. The extract is separated from undissolvedplant material and the water-miscible organic solvent removed, such asby distillation, rotary evaporation or other standard procedures forsolvent removal. The resultant extract containing water-soluble andnon-water soluble components may be dried to give an isoflavonecontaining extract, which may be formulated with one or morepharmaceutically acceptable carriers, excipients and/or auxiliaries. Theextract following distillation contains a small amount of oil whichincludes isoflavones in their aglucone form (referred to herein asisoflavones). This isoflavone-enriched oil, which may be dried, forexample in the presence of silica, may be formulated with one or morecarriers, excipients and/or auxiliaries to give an isoflavone containingextract. Alternatively, isoflavones may be further concentrated byaddition to the oil of a non-water soluble organic solvent such ashexane, heptane or octane having high solubility for oils but lowsolubility for isoflavones. The oil readily partitions into the organicsolvent, and an enriched isoflavone containing extract falls out ofsolution. The recovered extract may be dried, for example in an oven at50 C to about 12° C., and formulated with one or more pharmaceuticallyacceptable carriers, excipients and/or auxiliaries.

The clover (Trifolium spp.) may be red clover (T. pratense),subterranean clover (T. subterranean), white clover (T. repens), or anyclover related species, or chick pea variety. Preferably the clover isred clover (T. pratense) or subterranean clover (T. subterranean).

The compositions according to the present invention may include one ormore pharmaceutically acceptable carriers. Carriers are selected so asto be acceptable in the sense of being ingredients in the compositionand must not be deleterious to the patient. The carriers may be solid ora liquid, or both, and may be formulated with the extract as aunit-dose, for example a tablet, which may contain from 0.5% to 80% byweight of extract or up to 100% by weight to extract. Compositions maybe prepared by any of the well known techniques of pharmacy, for exampleadmixing the extract, optionally including excipients, diluents (forexample water) and auxiliaries as are well known in the pharmaceuticalfield.

The compositions according to the invention may include one or moreagents, such as vitamins (for example, Vitamin A, Vitamin B group,Vitamin C, Vitamin D, Vitamin E and Vitamin K), and minerals (forexample, magnesium, iron, zinc, calcium and manganese in the form ofpharmaceutically acceptable salts).

The compositions of the invention include those suitable for oral,rectal, optical, buccal (for example sublingual), parental (for examplesubcutaneous, intramuscular, intradermal and intravenous) andtransdermal administration. The most suitable route in any given casewill depend on the nature and severity of the condition being treatedand the state of the patient.

Compositions suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the extract; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchcompositions may be prepared by any suitable method of pharmacy, whichincludes the step of bringing into association the active compound, andone or more suitable carriers (which may contain one or more accessoryingredients as noted above). In general the compositions of theinvention are prepared by uniformly and intimately admixing the extractwith a liquid or finely divided solid carrier, or both, and then, ifnecessary, shaping the resulting mixture. For example, a tablet may beprepared by comprising or moulding a powder or granules containing theextract, optionally with one or more accessory ingredients. Compressedtables may be prepared by compressing in a suitable machine, theextracts in the form of a powder or granules optionally mixed with abinder, lubricant, inert diluents, and/or surface active/dispersingagent(s). Moulded tablets may be made by moulding, in a suitablemachine, the powdered compound moistened with an inert liquid binder.

Suitable carriers may be fillers, such as sugars, for example lactose,saccharose, mannitol or sorbitol, cellulose preparations and/or calciumphosphates, for example tricalcium phosphate or calcium hydrogenphosphate, and also binders, such as starch pastes using, for example,corn, wheat, rice or potato starch, gelatin, tragacanth, methylceulloseand/or polyvinylpyrrolidone, and, if desired, disintegrators, such asthe above-mentioned starches, also carboxymethyl starch, cross linkedpolyvinyl pyrrolidone, agar or alginic acid or a salt thereof, such assodium alginate. Excipients may be flow conditioners and lubricants, forexample silicic acid, talc, stearic acid or salts thereof, such asmagnesium or calcium stearate, and/or polyethylene glycol. Dragee coresare provided with suitable, optionally enteric, coatings, there beingused, inter alia, concentrated sugar solutions which may comprise gumarabic, talc, polyvinylpyrrolidone, polyethylene glycol and/or titaniumdioxide, or coating solutions in suitable organic solvents or solventmixtures, or, for the preparation of enteric coatings, solutions ofsuitable cellulose preparations, such as acetylcellulose phthalate orhydroxypropylmethylcellulose phthalate. Dyes or pigments may be added tothe tablets or dragee coatings, for example for identification purposesor to indicate different doses of active ingredients.

Other orally administrable pharmaceutical compositions are dry-filledcapsules made, for example, of gelatin, and soft, sealed capsules madeof gelatin and a plasticiser, such as glycerol or sorbitol. Thedry-filled capsules may comprise the extracts in the form of granules,for example in admixture with fillers, such as lactose, binders, such asstarches, and/or glicants, such as talc or magnesium stearate, and,where appropriate, stabilisers. In soft capsules, the extract ispreferably dissolved or suspended in suitable liquids, such as fattyoils, paraffin oil or liquid polyethylene glycols, to which stabilisersmay also be added.

Formulations suitable for buccal (sublingual) administration includelozenges comprising the extracts in a flavoured base, usually sucroseand acacia or tragacanth; and pastilles comprising the compound in aninert base such as gelatin and glycerin or sucrose and acacia.

Compositions of the present invention suitable for parenteraladministration conveniently comprise sterile aqueous preparations of theextracts, which preparations are preferably isotonic with the blood ofthe intended recipient. These preparations are preferably administeredintravenously, although administration may also be effected by means ofsubcutaneous, intramuscular, or intradermal injection. Suitablecompositions include water soluble extracts and also suspensions of theactive ingredient, such as corresponding oily injection suspensions,there being used suitable lipophilic solvents or vehicles, such as fattyoils, for example sesame oil, or synthetic fatty acid esters, forexample ethyl oleate or triglycerides, or aqueous injection suspensionscomprising viscosity-increasing substances, for example sodiumcarboxymethylcellulose, sorbitol and/or dextran, and, where appropriate,also stabilisers. As an example compositions may conveniently beprepared by admixing the extracts with water or a glycine buffer andrendering the resulting solution sterile and isotonic with the blood.Injectable formulations according to the invention may contain from 0.1%to 60% w/v of the extract and may, for example, be administered at arate of 0.1 ml/minute/kg.

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by admixing theextracts with one or more conventional solid carriers, for example cocoabutter, and then shaping the resulting mixture.

Compositions suitable for topical administration to the skin preferablytake the form of an ointment, cream, lotion, paste, gel, spray, aerosol,or oil. Carriers which may be used include petroleum jelly, lanoline,polyethylene glycols, alcohols, and a combination of two or morethereof.

The extract is generally present at a concentration of from 0.1% to 30%weight/weight, for example from 0.5% to 10% weight/weight. Theisoflavone ratio in the composition is preferably in the range ofbiochanin:formononetin from 2:1 to 8:1. Small or trace amounts ofdaidzein and biochanin may be present.

Compositions suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchesmay contain the extracts in an optionally buffered aqueous solution.

Compositions suitable for transdermal administration may also bedelivered by iontophoresis (see, for example, Pharmaceutical Research 3(6), 318 (1986)) and typically take the form of an optionally bufferedaqueous solution of the extracts. Such compositions may, for example,contain citrate or bis/tris buffer (pH 6) or ethanol/water, with forexample 0.05% to 30% w/w extract.

Compositions may be prepared in a manner, and in a form/amount as isconventionally practised. See for example, Goodman & Gillman, ThePharmalogical Basis of Therapeutics (7th Edition, 1985) and Remington'sPharmaceutical Science (Mack Publishing Company, 10th Edition), both ofwhich are incorporated herein by reference. Compositions may contain,for example, from 0.1 mg to 2 g extract, such as 0.1 mg to 200 mg.

The extracts may be in the form of a powder, a slurry, in aqueoussolution (for example containing a small amount of oil), particulateform, or dissolved in an organic solvent (such as methanol, ethanol,ethylacetate or dimethyl sulphoxide).

What constitutes an effective amount of the compositions of the presentinvention will depend upon a number of factors, such as specific mode ofadministration, the condition being treated, the condition of thepatient and the judgement of the health care giver. Examples of dosagesof extracts are about 0.1 mg to about 200 mg per day, such as in theorder of 1.5 mg/kg (body weight)/day.

Preferably the extract comprises biochanin as the principal isoflavone.In the context of this invention, it has been found that the preferredratio of biochanin to the other three main estrogenic isoflavonesembraces the various naturally-occurring forms of isoflavones includingtheir aglycone, glycoside, acetyl or malonyl forms.

This invention will now be described with reference to the followingnon-limiting examples.

EXAMPLE 1

A cultivar of red clover specially selected for its high biochanincontent (85% biochanin, 10% formononetin, 2.5% daidzein and 2.5%genistein) is harvested and snap-frozen within 4 hours by exposure toliquid nitrogen. The material can be stored in this form for up toseveral years. For extraction, the frozen material is crushed to a finepowder, thawed and placed in a fine gauze bag, which is immersed in asolution of 60% ethanol in water. Extraction is carried out at 90° C.for 24 hours. The supernatant is separated from the undissolved plantmaterial, and the solvent removed by distillation. The aqueous phasecontaining isoflavones in an aglucone form is extracted with an organicsolvent (either petroleum ether or hexane or acetyl acetate) to removeoils and other polar compounds. The solvent then is removed bydistillation and the aqueous phase taken to near-dryness by rotaryevaporation. The tar-like residue then is oven-dried. The final powdertypically contains 40-50% isoflavones in the ratio of biochanin:formononetin, daidzein and genistein of 5:1.

The ratio of biochanin to formononetin, daidzein and genistein may beadjusted from about 2:1 to 5:1. At this ratio the unexpected therapeuticeffects of the invention are observed. The biochanin to formononetin,daidzein and genistein can be easily adjusted by HPLC fractionation ofthe Example 1 material, extracting a red clover cultivar comprising theappropriate ratio of components (ie. from 2:1-5:1 biochanin toformononetin, daidzein and genistein), or by adjusting the concentrationof the extracting organic solvent (less organic solvent less extractedbiochanin).

EXAMPLE 2

The plasma and urinary profiles of isoflavones after acute and chronicadministration of 22 mg of isoflavones (genistein 0.5 g, daidzein 0.5mg, biochanin 26 g, and formononetin 14 mg) in fourteen subjects wasanalysed. The composition corresponds to the Promensil product(Registered Trade Mark, Novogen Research Pty Ltd, Australia). Venousplasma and urine were collected at intervals for 24 h after whichsubjects commenced taking two tablets each day for two weeks andrepeated plasma samples and urine were collected for a 48 h period afterthe last does. Plasma and urine isoflavones were assayed by HPLC.

After acute dosing all four isoflavones appeared rapidly in plasma andreached peak levels around 6 h. Although daidzein and genistein werepresent in much higher concentrations than their methylated precursors,indicating a rapid demethylation of these compounds, formononetin andbiochanin remained detectable in both plasma and urine at all times.This was a quite unexpected finding.

EXAMPLE 3

Pharmaceutical compositions are prepared from the extracts according tothe examples above.

1. The following composition is prepared in the form of a tablet:

-   -   40-60 mg of extract from Example 1    -   340 mg of a standard tablet inert carrier

This composition is tableted to provide a 380-400 mg tablet.

2. The following composition is prepared in the form of a capsule:

-   -   40-60 mg of extract of Example 1 or 2    -   190 mg of a standard pharmaceutical inert carrier        all contained in a non-toxic gelatin capsule.

The carriers referred to above include cellulose (microcrystalline),calcium hydrogen phosphate, soy polysacchardie, magnesium stearate andsilica-colloidal (anhydrous).

EXAMPLE 4

A 38-year old pre-menopausal woman with uterine fibroids suffered fromsevere and irregular menstrual bleeding. Ultrasound examination hadconfirmed the presence of a number of fibroids that were protrudingthrough the endometrium. She had been taking a soy supplement deliveringabout 45 mg of both genistein and daidzein daily for six months with noapparent change in her symptoms. Within three months of startingtreatment with red clover extract according to Example 3, her bleedingand menstrual pain became much less severe and her menstruation returnedessentially to normal. An ultrasound confirmed that the fibroids hadsignificantly reduced in size.

EXAMPLE 5

A 36-year old pre-menopausal woman had suffered endometriosis for over 5years with progressive worsening. A laparoscopic examination hadconfirmed the presence of extensive endometriosis lesions over herovaries and serosal surface of the uterus. For 1 year she had tried adietary change including eating tofu and soymilk on a regular basis,both foods known to contain high levels of daidzein and genistein. Thecondition continued to worsen and finally she was scheduled for surgeryfor laser burning of the islets of endometriosis tissue. She took a redclover extract according, to Example (3) for 3 months prior to surgery.At the time of surgery her symptoms (pelvic pain) had reducedconsiderably, but it was decided to still proceed with the surgery.Laparoscopy failed to reveal any evidence of endometriosis lesions. Sheremains symptom-free 9 months later with ongoing daily treatment withthe red clover extract.

EXAMPLE 6

A 44-year old woman had suffered ovarian cysts for 8 years, producingdysmennorhoea and low-grade pain. Various, treatments including hormonaltreatments had failed to improve the condition. After taking the redclover extract according to Example (3) for 3 months, her menstrualperiod returned to normal frequency and level of bleeding and the painsubsided. An ultrasound examination confirmed the absence of ovariancysts on either ovary. She remains well 6 months later with no evidenceof return of the cysts.

EXAMPLE 7

A 29-year old pre-menopausal woman had suffered endometrial hyperplasiafor 9 years. The condition caused symptoms of heavy, frequentmenstruation and menstrual cramps and pain. She had been treatedperiodically by curettage which had provided temporary relief only.After taking the red clover extract according to Example (3) for 4months, her menstruation returned to normal frequency, length, durationand level of bleeding.

EXAMPLE 8

A 19-year old pre-menopausal woman suffering from severe acne andcyclical mastalgia was given the red clover extract according to Example(3) for a period of two menstrual cycles. Her pain score diary showedsignificant improvement for breast pain and tenderness, and a noticableimprovement was seen in her acne.

EXAMPLE 9

A young woman, Mrs. M, with recurrent endometriosis, and suffered severepain and to irregular bleeding. Following daily treatment according toExample 4, her monthly pain and irregular bleeding were minimal, andprognosis for conception was improved. Improvements was seen shortlyafter treatment began.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”or the term “includes” or variations thereof, will be understood toimply the inclusion of a stated element or integer or group of elementsor integers but not the exclusion of any other element or integer orgroup of elements or integers. In this regard, in construing the claimscope, an embodiment where one or more features is added to any ofclaims is to be regarded as within the scope of the invention given thatthe essential features of the invention as claimed are included in suchan embodiment.

REFERENCES

-   1. Grodstein, F., Colditz, G. A., and Stampfer, M. J.    “Postmenopausal hormone use and colecystectomy in a large    prospective study” Obstetrics and Gynaecology: 1994: 83(1), 5-11.-   2. Akkad, A. A., Habiba, M. A., Ismail, N., Abrams, K., and    al-Azzawi, F. “Abnormal uterine bleeding on HRT: The impotace of    intrauterine structural abnormalities” Obstetrics and    Gynaecology, 1995. 86(3): 330-334.-   3. Sener, A. G., Seckin, N. C., Ozmien, S., Gokmen, O., Dou, N. and    Ekici, E. “The effects of HRT on uterine fibroids in postmenopausal    women” Fertility and Sterility, 1996. 65(2): 354-357.-   4. Namnoum, A. G., Hickman, T. N., Goodman, S. B., Gehlbach, D. L.,    and Rock, J. A. “Incidence of symptoms recurrence after hysterectomy    for endometriosis” Fertility and Sterility, 1995. 64(5): 898-902.-   5. Goh, J. T. and Hall, B. B. “Postmenopausal endometrioma and HRT”    Australia New Zealand of Obstetrics and Gynaecology, 1992. 32(4):    384-385.-   6. Sanchez-Guerrero, J., Liang, M. H., Karlson, E. W., Hunter, D. J.    and Colditz, G. A. “Postinenopausal estrogen therapy and the risk    for developing systemic lupus erythematosus” Annals of Internal    Medicine 1995. 122(6): 430-433.-   7. Troissi. R. J., Speizer, F. E., Willett, W. C., Trichopolous, D.    and Rosner, B. “Menopause, postmenopausal estrogen preparations ad    the risk of adult-onset asthma. A prospective cohort study” American    Journal of Respiratory and Critical Care Medicine. 1995, 152(1),    1183-1188.

1-18. (canceled)
 19. A method for the treatment, prophylaxis,amelioration or prevention of a condition selected from the groupconsisting of: uterine fibroids, polycystic ovarian disease, ovariancysts, cyclical acne, mastalgia, endometriosis and endometrialhyperplasia, said method comprising administration of anisoflavone-containing extract of clover or chick pea, or anisoflavone-containing extract of clover or chick pea comprisingprimarily biochanin and any one or more of the isoflavones from thegroup formononetin, daidzein and genistein at a ration from about 2:1 toabout 5:1
 20. The method according to claim 19, wherein theisoflavone-containing extract primarily comprising biochanin comprisesat least 65% biochanin by weight, the remainder comprising essentiallyformononetin and/or daidzein and/or genistein.
 21. The method accordingto claim 20, comprising 70 to 100% biochanin.
 22. The method accordingto claim 19, wherein the ratio of biochanin to one or more of theisoflavones from the group formononetin, daidzein and genistein is fromabout 7:3 to about 5:1.
 23. The method according to claim 19, whereinthe extract of clover or chick pea is a water/water-miscible organicsolvent extract.
 24. The method according to claim 19, wherein theclover is red clover.
 25. The method according to claim 19, wherein theisoflavone is in a glycoside, agylcone, acetyl or malonyl form.
 26. Amethod for the treatment, prophylaxis, amelioration or prevention of acondition selected from the group consisting of: uterine fibroids,polycystic ovarian disease, ovarian cysts, cyclical acne, mastalgia,endometriosis and endometrial hyperplasia, said method comprisingadministration of a composition comprising an isoflavone-containingextract of clover or chick pea, or an isoflavone-containing extract ofclover or chick pea comprising primarily biochanin, or a ratio ofbiochanin to one or more of the isoflavones from the group formononetin,daidzein and genistein in the range of from about 2:1 to about 5:1,optionally in association with one or more pharmaceutically acceptablecarriers, excipients, auxiliaries, and/or diluents.
 27. The methodaccording to claim 26, wherein the isoflavone-containing extractprimarily comprising biochanin comprises at least 65% biochanin byweight, the remainder comprising isoflavones from the groupformononetin, daidzein and genistein.
 28. The method according to claim27, comprising 70 to 100% biochanin.
 29. The method according to claim26, wherein the ratio of biochanin to one or more of the isoflavonesfrom the group formononetin, daidzein and genistein is in the range offrom about 7:3 to about 5:1.
 30. The method according to claim 26,wherein the isoflavone is in a glycoside, agylcone, acetyl or malonylform.
 31. The method according to claim 19, wherein the condition ispolycystic ovarian disease.
 32. The method according to claim 26,wherein the condition is polycystic ovarian disease.